Anti-stem cell factor antibodies and methods of use thereof in renal disease

ABSTRACT

The disclosure relates to methods of use in renal diseases and disorders of antibodies and antigen-binding fragments thereof that bind to Stem Cell Factor (SCF). The antibodies and antigen-binding fragments thereof specifically bind to SCF248 and are useful for treating inflammatory and fibrotic renal disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 62/900,927, filed on Sep. 16, 2019, the entire contentsof which are hereby incorporated by reference.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The content of the text file submitted electronically herewith isincorporated herein by reference in its entirety: A computer readableformat copy of the Sequence Listing (filename:OPSL_001_02WO_SeqList_ST25; date recorded: Sep. 16, 2020; file size: 58kb).

BACKGROUND

Inflammatory diseases are a major cause of morbidity and mortalityworldwide. Some types of chronic inflammation can lead to fibrosis,which is the formation or development of excess fibrous connectivetissue in an organ or tissue as a reparative or reactive process, asopposed to formation of fibrous tissue as a normal constituent of anorgan or tissue. Chronic inflammation as well as fibrosis can affectnearly all tissues and organ systems, and fibrotic tissue remodeling caninfluence cancer metastasis and accelerate chronic graft rejection intransplant recipients. Chronic inflammation of the kidney can lead tofibrotic diseases with a high rate of mortality.

Stem cell factor (SCF) and its receptor c-Kit are important factors ofthe perpetuation of chronic inflammation and in fibrotic diseases(El-Koraie, et al., Kidney Int. 60: 167 (2001); Powell, et al., Am. J.Physiol. 289: G2 (2005); El Kossi, et al., Am. J. Kidney Dis. 41: 785(2003); Powell, et al., Am. J. Physiol. 277: C183 (1999) Ding et al JPathol. 2013 June; 230(2):205-14., Berlin et al Lab Invest. 2006 June;86(6):557-65, Rasky et al Am J Physiol Lung Cell Mol Physiol. 2020 Jan.1; 318(1):L200-L211). c-Kit is a type III receptor-tyrosine kinase thatis present in many cell types (Orr-Urtreger et al., Development 109: 911(1990). Immune cells such as mast cells, eosinophils, and innatelymphoid cells 2 and 3 (ILC2 and ILC3) are all c-Kit+ cells that maydrive the chronic inflammatory process, depending on the disease andorgan involved. Upon initiation of an inflammatory response, variousmediators, including SCF, activate c-Kit+ immune cells, which in turnproduce cytokines that cause fibroblasts to become activatedmyofibroblasts. Myofibroblasts secrete extracellular matrix proteins,collagen, and fibronectin, resulting in fibrosis of tissue. Activatedmyofibroblasts, activated epithelia, endothelia, macrophages,eosinophils, mast cells, monocytes, and other cells also express SCF onthe cell surface, which activates more c-Kit+ immune cells, resulting inmore cytokine release and perpetuating the inflammation.

There is a need in the art for more efficient and more specifictreatments for inflammatory and fibrotic diseases of the kidney. Thepresent disclosure addresses this and other needs.

SUMMARY OF THE DISCLOSURE

In one aspect, the present disclosure provides methods of treating renaldiseases and disorders, the methods comprising administering to apatient having a renal disease or disorder an antibody or fragmentthereof that specifically binds to stem cell factor (SCF). Inembodiments, the renal disease or disorder is an inflammatory renaldisease, a fibrotic renal disease, and/or a tissue remodeling renaldisease. In embodiments, the antibodies and fragments thereof for use inthe methods provided herein specifically bind to the SCF isoform SCF248.In some embodiments, the antibodies and fragments thereof for use in themethods provided herein comprise heavy chain complementarity determiningregions (CDRs), wherein heavy chain CDR1 CDR2, and CDR3 comprise SEQ IDNOs: 1, 2, and 3, respectively. In some embodiments, the antibodies andfragments thereof for use in the methods provided herein comprise lightchain CDRs, wherein the light chain CDR1 CDR2, and CDR3 comprise SEQ IDNOs: 4, 5, and 6, respectively. In some embodiments, the antibodies andfragments thereof for use in the methods provided herein comprise heavychain CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 1, 37, and 3,respectively. In some embodiments, the antibodies and fragments thereofcomprise a heavy chain variable region comprising at least 80%, at least85%, at least 90%, at least 95%, or at least 99% identity to a sequenceselected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11, and12. In some embodiments, the antibodies and fragments thereof comprise alight chain variable region comprising at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% identity to a sequence selectedfrom the group consisting of SEQ ID NOs: 13, 14, 15, 16, and 17. In someembodiments, the antibodies and fragments thereof comprise a heavy chainvariable region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 7, 8, 9, 10, 11, and 12, and a lightchain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 13, 14, 15, 16, and 17.

In some embodiments, the present disclosure provides methods fortreating inflammatory and/or fibrotic diseases of the kidney, comprisingadministering to a subject an antibody or fragment thereof thatcomprises a heavy chain variable region amino acid sequence according toSEQ ID NO: 7 and a light chain variable region amino acid sequenceaccording to SEQ ID NO: 16. In some embodiments, the antibody orfragment thereof of claim 1, wherein the antibody or fragment thereofcomprises a heavy chain variable region amino acid sequence according toSEQ ID NO: 8 and a light chain variable region amino acid sequenceaccording to SEQ ID NO: 16. In some embodiments, the antibody orfragment thereof comprises a heavy chain variable region amino acidsequence according to SEQ ID NO: 9 and a light chain variable regionamino acid sequence according to SEQ ID NO: 16. In some embodiments, theantibody or fragment thereof comprises a heavy chain variable regionamino acid sequence according to SEQ ID NO: 10 and a light chainvariable region amino acid sequence according to SEQ ID NO: 16. In someembodiments, the antibody or fragment thereof comprises a heavy chainvariable region amino acid sequence according to SEQ ID NO: 11 and alight chain variable region amino acid sequence according to SEQ ID NO:16. In some embodiments, the antibody or fragment thereof comprises aheavy chain variable region amino acid sequence according to SEQ ID NO:12 and a light chain variable region amino acid sequence according toSEQ ID NO: 16.

In some embodiments, the antibody or fragment thereof is humanized. Insome embodiments, the antibody is a monoclonal antibody. In someembodiments, the antibody comprises a human IgG1 domain or a human IgG4domain. In some embodiments, the antibody is an antigen bindingfragment, wherein the fragment is selected from a Fab, F(ab′)2, Fab′,scFv, and single domain antibody (sdAb).

In some embodiments, the antibody or fragment thereof blocks theinteraction between SCF (e.g. SCF248) and c-Kit. In some embodiments,the antibody specifically binds to SCF248. In some embodiments, theantibody does not bind to SCF220. In some embodiments, the antibodyprevents the interaction of SCF248 and c-kit by causing theinternalization of SCF, making it unavailable on the cell surface.

In one aspect, the present disclosure provides pharmaceuticalcompositions comprising the antibody or fragment thereof providedherein. In some embodiments, the pharmaceutical composition comprises apharmaceutically acceptable carrier, diluent or excipient.

In some embodiments, the present disclosure provides isolated nucleicacid molecules encoding the antibody or fragment thereof providedherein. In some embodiments, the present disclosure provides anexpression vector comprising the nucleic acid encoding the antibody orfragment thereof. In some embodiments, the present disclosure provides arecombinant host cell comprising the expression vector.

In one aspect, the present disclosure provides methods for making anantibody that specifically binds to stem cell factor isoform 248(SCF248), the method comprising immunizing a host animal with a peptidecomprising SEQ ID NO: 30 (ASSLRNDSSSSNRKAKNPPGD) or a fragment thereof,and obtaining an antibody from the immunized host animal. In someembodiments, the host animal is not a human. In some embodiments, thefragment of SEQ ID NO: 30 comprises at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, or 20 contiguous amino acids of SEQ ID NO: 30. In someembodiments, the fragment of SEQ ID NO: 30 comprises 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids of SEQID NO: 30. In some embodiments, the N-terminal amino acid of thefragment of SEQ ID NO: 30 is the alanine at position 1 at the N-terminusof SEQ ID NO: 30. In some embodiments, the method comprises immunizingthe host animal with a peptide consisting of SEQ ID NO: 30. In someembodiments, the antibody from the immunized host animal is obtainedfrom an immune cell isolated from the host animal. In some embodiments,the method further comprises generating a hybridoma using the immunecell. Thus, in some embodiments, the present disclosure provideshybridomas that produce monoclonal antibodies described herein.

In one aspect, the present disclosure provides an antibody or fragmentthereof that specifically binds to SCF248, wherein the antibody orfragment thereof binds to an epitope comprising at least 8, at least 9,at least 10, at least 11, at least 12, or at least 13 contiguous aminoacids of SEQ ID NO: 33, wherein the antibody inhibits the interaction ofSCF248 with c-Kit. In further embodiments, the epitope comprises SEQ IDNO: 33 or SEQ ID NO: 36. In yet further embodiments, the epitopeconsists of SEQ ID NO: 33 or SEQ ID NO: 36.

In one aspect, the present disclosure provides compositions and methodsfor inhibiting the interaction between SCF and c-Kit. C-kit is expressedon immune cells, hematopoietic stem cells, and some structural cells.C-kit's ligand SCF248 can be upregulated on myofibroblasts, activatedepithelia, endothelia, macrophages, eosinophils, mast cells, monocytes,and others. In some embodiments, the compositions and methodsspecifically inhibit the interaction between SCF248 and c-Kit. Forexample, in some embodiments, the compositions and methods specificallyinhibit the interaction between SCF248 on myofibroblasts and c-Kit onimmune cells. As another example, in some embodiments, the compositionsand methods provided herein specifically inhibit the interaction betweenSCF248 on myofibrobalsts, activated epithelia, endothelia, macrophages,eosinophils, mast cells, and/or monocytes; with c-Kit on immune cellsand/or structural cells. In some embodiments, the methods comprisecontacting SCF248 on myofibroblasts with an antibody or fragment thereofprovided herein. In some embodiments, the antibody or fragment thereofprovided herein blocks binding of SCF248 to c-Kit. In some embodiments,the blocking is via steric hindrance. In some embodiments, the antibodyor fragment thereof provided herein internalizes SCF248.

In some embodiments, the present disclosure provides methods forinhibiting inflammation in a subject in need thereof, the methodcomprising administering to the subject an antibody or fragment thereofprovided herein. In some embodiments, the present disclosure providesmethods for inhibiting an inflammatory disease in a subject in needthereof, the method comprising administering to the subject an antibodyor fragment thereof provided herein. In further embodiments, theinflammatory disease is a chronic inflammatory disease. In someembodiments, the present disclosure provides methods for treatinginflammation and/or a chronic inflammatory disease in a subject in needthereof, the method comprising administering to the subject an antibodyor fragment thereof provided herein.

In some embodiments, the present disclosure provides methods forinhibiting fibrosis in a subject in need thereof, the method comprisingadministering to the subject an antibody or fragment thereof providedherein. In some embodiments, the present disclosure provides methods fortreating a fibrotic disease in a subject in need thereof, the methodcomprising administering to the subject an antibody or fragment thereofprovided herein. In embodiments, the method further comprisesadministering one or more additional therapy and/or therapeutic agent.

In some embodiments, the inflammatory renal disease or fibrotic renaldisease is selected from the group consisting of renal fibrosis,Interstitial Fibrosis and Tubular Atrophy (IFTA) of the kidney, chronickidney disease, end stage renal disease (ESRD), glomerulonephritis,chronic renal allograft rejection, nephrogenic systemic fibrosis, andnephropathy (e.g., IgA nephropathy, focal segmental glomerulosclerosis,rapidly progressive glomerulonephritis, crescentic glomerulonephritis,lupus nephritis, hypertensive nephropathy, or diabetic nephropathy).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic overview of the tissue injury/inflammatorydisease process.

FIG. 2 shows an exemplary mechanism of an anti-SCF248 antibody of theinstant disclosure, 5H10. The 5H10 antibody is referred to in the figureas “OpSCF”.

FIG. 3 shows the isoforms of SCF, SCF220 and SCF 248; and the monomericcleaved extracellular domain, SCF165. SCF165 is released upon cleavageof SCF248 at its cleavage site within the Exon 6 region.

FIG. 4A is a set of histograms showing the binding of murine 5H10antibody to control cells that do not expression SCF (left panel), cellsthat express SCF220 but not SCF248 (middle panel), and cells thatexpress SCF248 but not SCF220 (right panel).

FIG. 4B shows the binding of murine 5H10 antibody to the 165 amino acidcleaved SCF extracellular domain (ECD) versus the complete 194 aminoacid SCF ECD.

FIG. 5 shows the Mean Fluorescence Intensity (MFI) as measured by flowcytometry after contacting cultured human IPF myofibroblasts with pHrodored-labeled 2G8, 5H10, or control IgG antibodies.

FIG. 6 shows the activation of the PI3K/AKT pathway and the MEK/ERKpathway of c-kit signaling after contacting eosinophils with anSCF248-expressing cells in the presence of 5H10 antibody or IgG control.5H10 antibody significantly reduced activation of both pathways.

FIGS. 7A and 7B shows binding of 5H10 humanized variants at differentantibody concentrations by flow cytometry to Sl/Sl4 hSCF248 cells. InFIG. 7A, the indicated VH is paired with VK3. In FIG. 7B, the 5H10antibody shown is VH1/VK3.

FIG. 8A-8C show the change in mRNA level of the CCL11 (FIG. 8A),Collagen 1A1 (FIG. 8B), fibronectin (FIG. 8C), or collagen 3 (FIG. 8D)after preincubation of human IPF myofibroblasts (Mfb) with a positivecontrol (irrelevant antibody) or the antibody indicated under each barin the figure. The murine parent antibody is indicated as “5H10” in thefigure; humanized 5H10 antibodies VH1/VK3, VH2/VK3, VH3/VK3, VH4/VK3,and VH5/VK3 were also tested as shown. Antibody concentrations testedwere 1 μg/mL or 10 μg/mL.

FIG. 9A-FIG. 9C shows the correlation between SCF248 mRNA and glomerularfiltration rate (FIG. 9A), interstitial fibrosis (FIG. 9B), andpercentage of mononuclear white blood cells in the kidney biopsy (FIG.9C) in patients with focal segmental glomerulosclerosis (FSGS).

FIG. 10 shows that plasma levels of SCF165 (and its derivatives) areinversely correlated with estimated glomerular filtration rate (eGFR) inpatients with chronic kidney disease.

FIG. 11 shows that plasma levels of SCF165 (and its derivatives) areinversely correlated with the urinary albumin/creatinine ratio (UACR) inpatients with chronic kidney disease.

FIG. 12A-FIG. 12C shows immunohistochemistry staining for SCF248 in thetubular and mesangial areas of a human glomerulonephritis kidney biopsy.FIG. 12A shows staining with control IgG; FIGS. 12B and 12C showstrongly positive staining for SCF348 in the tubule-interstitium.

FIG. 13A-FIG. 13C shows immunohistochemistry staining for mast celltryptase in a healthy kidney (FIG. 13A) and in the kidneys of patientswith diabetic nephropathy and IgA nephropathy (FIGS. 13B and 13C,respectively).

FIG. 14A-FIG. 14B shows the effect of murine 5H10 (referred to as OpSCFin the figure) treatment on survival (FIG. 14A) and kidney weight (FIG.14B) in a TGFβ mouse model of CKD.

FIG. 15A-FIG. 15C shows the effect of murine 5H10 treatment onglomerular volume (FIG. 15A), mesangial volume (FIG. 15B), and podocytedensity (FIG. 15C) kidney weight in a TGFβ mouse model of CKD.

FIG. 16A-16F show RNA sequencing demonstrating statistically significantdecreases in several fibrillary matrix proteins. FIG. 16A: Collagen Type3 alpha 1 chain; FIG. 16B: Collagen Type 6 alpha 3 chain; FIG. 16C:Collagen Type XV alpha 1 chain; FIG. 16D: Fibronectin Type III Domaincontaining 1; FIG. 16E: Fibulin 1; FIG. 16F: Microfibril-associatedprotein 4.

DETAILED DESCRIPTION

Stem Cell Factor (SCF) is a key mediator of acute and chronicinflammation, fibrotic diseases, and tissue remodeling diseases. Theinteraction of SCF with c-Kit on immune cells initiates and perpetuatesinflammation and fibrosis. The present disclosure provides compositionsand methods for treating inflammatory and fibrotic renal diseases byinhibiting the interaction of SCF with c-Kit. Thus, the presentdisclosure provides methods for treating chronic renal inflammatorydiseases and fibrotic renal diseases such as chronic kidney disease. Themethods comprise administering an antibody or fragment thereof thatspecifically binds to SCF to a patient suffering from an inflammatoryand/or fibrotic renal disease. In embodiments, the antibodies andfragments thereof provided herein specifically bind to SCF248 and do notbind to SCF220. Thus, the present disclosure provides specific,effective methods for inhibiting inflammation and fibrosis in renaldiseases and disorders, and treating inflammatory renal diseases andfibrotic renal diseases.

Definitions

As used herein, the term “antibody” refers to a binding protein havingat least one antigen binding domain. The antibodies and fragmentsthereof of the present invention may be whole antibodies or any fragmentthereof. Thus, the antibodies and fragments of the invention includemonoclonal antibodies or fragments thereof and antibody variants orfragments thereof, as well as immunoconjugates. Antigen bindingfragments include Fab fragments, Fab′ fragments, F(ab′)2 fragments,bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, singlechain Fv proteins (“scFv”), bis-scFv, (scFv)2, minibodies, diabodies,triabodies, tetrabodies, disulfide stabilized Fv proteins (“dsFv”),single-domain antibodies (sdAb, nanobody), heavy-chain only antibodies(e.g., camelid VHH, camelid nanobody, shark Ig NAR), and portions offull length antibodies responsible for antigen binding. An isolatedantibody or antigen binding fragment thereof is one which has beenidentified and separated and/or recovered from a component of itsnatural environment.

In some embodiments, the antibodies and antigen binding fragmentsthereof are isolated antibodies and fragments thereof, Thus, the presentinvention provides isolated antibodies and antigen binding fragmentsthereof, and nucleic acids encoding such antibodies and fragments, aswell as compositions comprising such isolated antibodies, fragments, andnucleic acids. The term “isolated” refers to a compound of interest(e.g., an antibody or nucleic acid) that has been separated from itsnatural environment. The present invention further providespharmaceutical compositions comprising the isolated antibodies orfragments thereof, or nucleic acids encoding such antibodies orfragments, and further comprising one or more pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers include, forexample, excipients, diluents, encapsulating materials, fillers,buffers, or other agents.

As used herein, the term “derived” when used to refer to a molecule orpolypeptide relative to a reference antibody or other binding protein,means a molecule or polypeptide that is specific for, and capable ofbinding to, the same epitope as the reference antibody or other bindingprotein.

As used herein, the phrase “specific for” may mean that the antibodydoes not bind to the target due to only non-specific interactions, andthis property can be determined by comparison to an isotype control orsimilar. Specific binding does not necessarily require, although it mayinclude, exclusive binding to a single target. In embodiments, theantibodies provided herein specifically bind to SCF248, and do not bindSCF220.

The term “host cell” means a cell that has been transformed, or iscapable of being transformed, with a nucleic acid sequence and therebyexpresses a gene of interest. The term includes the progeny of theparent cell, whether or not the progeny is identical in morphology or ingenetic make-up to the original parent cell, so long as the gene ofinterest is present.

A “variant” of a polypeptide (e.g., an antigen binding protein, or anantibody) comprises an amino acid sequence wherein one or more aminoacid residues are inserted into, deleted from and/or substituted intothe amino acid sequence relative to another polypeptide sequence.Variants include antibodies and fragments thereof that have a recitedpercent identity to an antibody or fragment provided herein or to anantibody or fragment having a recited DNA or amino acid sequence.

The term “identity” refers to a relationship between the sequences oftwo or more polypeptide molecules or two or more nucleic acid molecules,as determined by aligning and comparing the sequences. “Percentidentity,” “percent homology,” “sequence identity,” or “sequencehomology” and the like mean the percent of identical residues betweenthe amino acids or nucleotides in the compared molecules and iscalculated based on the size of the smallest of the molecules beingcompared. For these calculations, gaps in alignments (if any) arepreferably addressed by a particular mathematical model or computerprogram (i.e., an “algorithm”). Methods that can be used to calculatethe identity of the aligned nucleic acids or polypeptides include thosedescribed in Computational Molecular Biology, (Lesk, A. M., ed.), 1988,New York: Oxford University Press; Biocomputing Informatics and GenomeProjects, (Smith, D. W., ed.), 1993, New York: Academic Press; ComputerAnalysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G.,eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, SequenceAnalysis in Molecular Biology, New York: Academic Press; SequenceAnalysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York:M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math.48:1073. In calculating percent identity, the sequences being comparedare typically aligned in a way that gives the largest match between thesequences.

The term “light chain” includes a full-length light chain and fragmentsthereof having sufficient variable region sequence to confer bindingspecificity. A full-length light chain includes a variable region domainand a constant region domain. The variable region domain of the lightchain is at the amino-terminus of the polypeptide. Light chains includekappa chains and lambda chains.

The term “heavy chain” includes a full-length heavy chain and fragmentsthereof having sufficient variable region sequence to confer bindingspecificity. A full-length heavy chain includes a variable regiondomain, three constant region domains, C_(H)1, C_(H)2, and C_(H)3. Thevariable heavy domain is at the amino-terminus of the polypeptide, andthe C_(H) domains are at the carboxyl-terminus, with the C_(H)3 beingclosest to the carboxy-terminus of the polypeptide. Heavy chains can beof any isotype, including IgG (including IgG1, IgG2, IgG3 and IgG4subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE. The term“isotype” refers to the antibody class encoded by the heavy chainconstant region genes. In some embodiments, the antibodies providedherein have an IgG4 heavy chain, or an IgG4 heavy chain comprisingcertain amino acid mutations. For example, in some embodiments, the IgG4comprises a mutation at position 228 (EU numbering scheme, Kabat et al.Sequence of proteins of immunologic interest, 5th ed Bethesda, Md., NIH1991) to inhibit Fab arm exchange. For example, in some embodiments, theIgG4 heavy chain is an IgG4 S228P heavy chain. In some embodiments, theheavy chain comprises one or more amino acid mutations that reducebinding to Fc receptors, and thereby reduce or eliminate effectorfunction of the antibody. For example, the heavy chain may comprisemutations at one or more of positions 233, 234, 235, 236, 237, 265, 309,331, and 409 (EU numbering).

The term “variable region” or “variable domain” refers to a portion ofthe light and/or heavy chains of an antibody, typically includingapproximately the amino-terminal 120 to 130 amino acids in the heavychain and about 100 to 110 amino terminal amino acids in the lightchain. In certain embodiments, variable regions of different antibodiesdiffer extensively in amino acid sequence even among antibodies of thesame species. The variable region of an antibody typically determinesspecificity of a particular antibody for its target. The term “target,”as used herein, refers to a molecule or a portion of a molecule capableof being bound by an antigen binding protein. In certain embodiments, atarget can have one or more epitopes. In certain embodiments, a targetis an antigen. The use of “antigen” in the phrase “antigen bindingprotein” simply denotes that the protein sequence that comprises theantigen can be bound by an antibody. In this context, it does notrequire that the protein be foreign or that it be capable of inducing animmune response.

The term “epitope” includes any determinant capable being bound by anantigen binding protein, such as an antibody or to a T-cell receptor. Anepitope is a region of an antigen that is bound by an antigen bindingprotein that targets that antigen, and when the antigen is a protein,includes specific amino acids that directly contact the antigen bindingprotein. Most often, epitopes reside on proteins, but in some instancescan reside on other kinds of molecules, such as nucleic acids. Epitopedeterminants can include chemically active surface groupings ofmolecules such as amino acids, sugar side chains, phosphoryl or sulfonylgroups, and can have specific three dimensional structuralcharacteristics, and/or specific charge characteristics. Generally,antibodies specific for a particular target antigen will preferentiallyrecognize an epitope on the target antigen in a complex mixture ofproteins and/or macromolecules. Antibody epitopes may be linear orconformational. In embodiments, the epitope provided herein is a linearepitope.

The use of the singular includes the plural unless specifically statedotherwise. The word “a” or “an” means “at least one” unless specificallystated otherwise. The use of “or” means “and/or” unless statedotherwise. The meaning of the phrase “at least one” is equivalent to themeaning of the phrase “one or more.” Furthermore, the use of the term“including,” as well as other forms, such as “includes” and “included,”is not limiting. Also, terms such as “element” or “component” encompassboth elements or components comprising one unit and elements orcomponents comprising more than one unit unless specifically statedotherwise. As used herein, the term “about” refers to an amount more orless than the stated parameter value, for example plus or minus five orten percent of the object that “about” modifies, or as one of skill inthe art would recognize from the context (e.g., approximately 50% of theinterval between values). The term “about” also includes the valuereferenced.

Stem Cell Factor

In humans, there are at least two forms of SCF, which have differentstructures and activities. SCF220 functions in several homeostaticfunctions, including hematopoiesis and spermatogenesis and is found inbone marrow, testis, and other tissues and organs. SCF220 is slowlycleavable and sometimes called “membrane SCF.” In contrast, SCF248 israpidly cleavable and comprises a cleavage site in exon 6, locatedbetween the N-terminal c-kit binding domain and the transmembranedomain. SCF248 may be referred to as “soluble SCF”. Exon 6 is excludedfrom SCF220 via alternative splicing, and SCF220 thus lacks thiscleavage site. A monomeric, extracellular domain (SCF165) is thecleavage product and serves as a biomarker in plasma for chronicinflammatory diseases. Plasma may also contain detectable levels of SCFextracellular domain that comes from SCF220, but the majority ofdetectable extracellular domain is expected to be SCF165. SCF248 is theisoform found on myofibroblasts, activated epithelial cells, and othercells, which activates immune cells during inflammation and contributesto perpetuation of fibrosis. More specifically, SCF248 binds to c-Kit onimmune cells, initiating production of cytokines that activatefibroblasts to become myofibroblasts, which secrete extracellular matrixproteins, collagen, and fibronectin. The activated myofibroblasts aswell as activated epithelia, endothelia, macrophages, eosinophils, mastcells, monocytes, and other cells also express SCF on the cell surface,activating more c-Kit+ immune cells, resulting in further cytokinerelease and immune activation and fibrotic responses.

The antibodies and antigen-binding fragments thereof disclosed hereinare specific for SCF. In some embodiments, the antibodies and fragmentsthereof are specific for human SCF. In some embodiments, the antibodiesand fragments thereof are specific for SCF248. In some embodiments, theantibodies bind SCF248 and do not bind other isoforms of SCF. In someembodiments, the antibodies bind SCF248 and do not bind to SCF220. Insome embodiments, the present disclosure provides methods for making anantibody or fragment thereof that is specific for SCF248. Exemplaryantibodies and fragments that are specific for SCF248, as well asmethods for making and using the antibodies and fragments, are providedin the present disclosure. In some embodiments, the antibodies andfragments thereof provided herein breaks the positive feedback loopbetween SCF248 expressed on various cell types and cKit+ immune cells,by binding to SCF248 and blocking the interaction between SCF248 andc-Kit.

Antibodies and Fragments

The present disclosure provides antibodies, including monoclonalantibodies, and fragments thereof. The antibody fragments providedherein that are specific for SCF (e.g., SCF248) are sometimes referredto herein as antigen-binding fragments, meaning that they comprise theportion of the parent antibody that is capable of binding the targetantigen (SCF, e.g., SCF248). “Antibody fragment,” “antigen bindingfragment” and the like are used interchangeably herein. Examples ofantibody fragments include Fab fragments, Fab′ fragments, F(ab)′fragments, Fv fragments, isolated CDR regions, bispecific Fab dimers(Fab2), trispecific Fab trimers (Fab3), single chain Fv proteins(“scFv”), bis-scFv, (scFv)2, minibodies, diabodies, triabodies,tetrabodies, disulfide stabilized Fv proteins (“dsFv”), single-domainantibodies (sdAb, nanobody), heavy-chain only antibodies (e.g., camelidVHH, camelid nanobody, shark Ig NAR), and portions of full lengthantibodies responsible for antigen binding.

A “Fab fragment” comprises one light chain and the C_(H)1 and variableregions of one heavy chain. The heavy chain of a Fab molecule cannotform a disulfide bond with another heavy chain molecule. A “Fab′fragment” comprises one light chain and a portion of one heavy chainthat contains the VH domain and the CHI domain and also the regionbetween the C_(H)1 and C_(H)2 domains, such that an interchain disulfidebond can be formed between the two heavy chains of two Fab′ fragments toform an F(ab′)₂ molecule. A “F(ab′)₂ fragment” contains two light chainsand two heavy chains containing a portion of the constant region betweenthe C_(H)1 and C_(H)2 domains, such that an interchain disulfide bond isformed between the two heavy chains. A F(ab′)₂fragment thus is composedof two Fab′ fragments that are held together by a disulfide bond betweenthe two heavy chains. A “Fv fragment” comprises the variable regionsfrom both the heavy and light chains, but lacks the constant regions.“scFvs” are Fv molecules in which the heavy and light chain variableregions have been connected by a flexible linker to form a singlepolypeptide chain, which forms an antigen binding region.

In some aspects, the antibodies and fragments thereof provided hereinare defined by their complementary determining regions (CDRs). CDRs arepart of the variable chains in antibodies; each of the light and heavychain variable regions comprises three CDRs, CDR1, CDR2, and CDR3. TheCDRs of an antibody determine antigen specificity. In certainembodiments, definitive delineation of a CDR and identification ofresidues comprising the binding site of an antibody is accomplished bysolving the structure of the antibody and/or solving the structure ofthe antibody-ligand complex. In certain embodiments, that can beaccomplished by any of a variety of techniques known to those skilled inthe art, such as X-ray crystallography. In certain embodiments, variousmethods of analysis can be employed to identify or approximate the CDRregions. Examples of such methods include, but are not limited to, theKabat definition, the Chothia definition, the AbM definition and thecontact definition.

The Kabat definition is a standard for numbering the residues in anantibody and is typically used to identify CDR regions. See, e.g.,Johnson & Wu, Nucleic Acids Res., 28: 214-8 (2000). The Chothiadefinition is similar to the Kabat definition, but the Chothiadefinition takes into account positions of certain structural loopregions. See, e.g., Chothia et al., J. Mol. Biol., 196: 901-17 (1986);Chothia et al., Nature, 342: 877-83 (1989). The AbM definition uses anintegrated suite of computer programs produced by Oxford Molecular Groupthat model antibody structure. See, e.g., Martin et al., Proc Natl AcadSci (USA), 86:9268-9272 (1989); “AbM™, A Computer Program for ModelingVariable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. TheAbM definition models the tertiary structure of an antibody from primarysequence using a combination of knowledge databases and ab initiomethods, such as those described by Samudrala et al., “Ab Initio ProteinStructure Prediction Using a Combined Hierarchical Approach,” inPROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999). Thecontact definition is based on an analysis of the available complexcrystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45(1996).

Antibodies and fragments thereof may also include recombinantpolypeptides, fusion proteins, and bi-specific antibodies. The anti-SCFantibodies and fragments thereof disclosed herein may be of an IgG1,IgG2, IgG3, or IgG4 isotype. In one embodiment, the anti-SCF antibodiesand fragments thereof disclosed herein are of an IgG1 or an IgG4isotype. The anti-SCF antibodies and fragments thereof of the presentinvention may be derived from any species including, but not limited to,mouse, rat, rabbit, primate, llama, camel, goat, shark, chicken, andhuman. The SCF antibodies and fragments thereof may be chimeric,humanized, or fully human antibodies. In one embodiment, the anti-SCFantibodies are murine antibodies. In another embodiment, the anti-SCFantibodies are chimeric antibodies. In a further embodiment, thechimeric antibodies are mouse-human chimeric antibodies. In anotherembodiment, the antibodies are derived from mice and are humanized.

A “chimeric antibody” is an antibody having at least a portion of theheavy chain variable region and at least a portion of the light chainvariable region derived from one species; and at least a portion of aconstant region derived from another species. For example, in oneembodiment, a chimeric antibody may comprise murine variable regions anda human constant region.

A “humanized antibody” is an antibody containing complementaritydetermining regions (CDRs) that are derived from a non-human antibody;and framework regions as well as constant regions that are derived froma human antibody. For example, the anti-SCF antibodies provided hereinmay comprise CDRs derived from one or more murine antibodies and humanframework and constant regions. Thus, in one embodiment, the humanizedantibody provided herein binds to the same epitope on SCF as the murineantibody from which the antibody's CDRs are derived.

In some embodiments, the antibodies and fragments thereof providedherein comprise a heavy and light chain, each of which comprises threeCDRs. The amino acid sequences of exemplary heavy chain CDR1, CDR2, andCDR3 (HCDR1, HCDR2, and HCDR3, respectively) and light chain CDR1, CDR2,and CDR3 (LCDR1, LCDR2, and LCDR3, respectively) are provided below inTable 1. Table 1 also provides the amino acid sequences of exemplaryheavy and light chain variable regions. In some embodiments, the presentdisclosure provides antibodies referred to herein as “5H10” and “2G8”.The heavy chain variable regions of humanized 5H10 or 2G8 are referredto herein as VH1, VH2, VH3, VH4, and VH5. 5H10 VH0 is the variable heavychain of the murine parent antibody generated via the methods describedherein. VH1, VH2, VH3, VH4, and VH5 are each humanized heavy chainvariable regions derived from 5H10 VH0 or 2G8 VH0. The 5H10 antibodycomprises a kappa light chain. The murine parent antibody variable lightchain is referred to herein as 5H10 VK0. VK1, VK2, VK3, and VK4 are eachhumanized light chain variable regions derived from VK0. The 2G8antibody comprises a lambda light chain. The murine parent antibodyvariable light chain is referred to herein as 2G8 VL0. VL1, VL2, VL3,and VL4 are each humanized light chain variable regions derived fromVL0.

TABLE 1 Exemplary anti-SCF antibody sequences SEQ ID NO Description 15H10 Heavy SYWMN chain CDR1 2 5H10 Heavy QIYPGDGDTHYNGKFKG chain CDR2 35H10 Heavy SNWVGSY chain CDR3 4 5H10 Light KSSQSLLESDGKTYLN chain CDR1 55H10 Light LVSRLDS chain CDR2 6 5H10 Light WQGTHLPQT chain CDR3 75H10 Heavy QVQLQQSGAELVRPGSSVKISCKSSGYAFSSYWMNWVKQRPGQG chain variableLEWIGQIYPGDGDTHYNGKFKGKATLTADKSSSTAYMQLSRLTS region VH0EDSAVYFCSSSNWVGSYWGQGTLVTVSA (murine parent) 8 5H10 HeavyQVQLVQSGAELKKPGSSVKISCKSSGYAFSSYWMNWVKQRPGQG chain variableLEWIGQIYPGDGDTHYNGKFKGKATLTADKSTSTAYMELSSLTS region VH1EDSAVYFCSSSNWVGSYWGQGTLVTVSS (humanized) 9 5H10 HeavyQVQLVQSGAEVKKPGSSVKISCKSSGYAFSSYWMNWVKQRPGQG chain variableLEWIGQIYPGDGDTHYNGKFKGKATLTADKSTSTAYMELSSLRS region VH2EDTAVYFCSSSNWVGSYWGQGTLVTVSS (humanized) 10 5H10 HeavyQVQLVQSGAEVKKPGSSVKVSCKSSGYAFSSYWMNWVRQRPGQG chain variableLEWIGQIYPGDGDTHYNGKFKGKATLTADKSTSTAYMELSSLRS region VH3EDTAVYFCSSSNWVGSYWGQGTLVTVSS (humanized) 11 5H10 HeavyQVQLVQSGAEVKKPGSSVKVSCKSSGYAFSSYWMNWVRQRPGQG chain variableLEWIGQIYPGDGDTHYNGKFKGRVTITADKSTSTAYMELSSLRS region VH4EDTAVYFCSSSNWVGSYWGQGTLVTVSS (humanized) 12 5H10 HeavyQVQLVQSGAEVKKPGSSVKVSCKSSGYAFSSYWMNWVRQRPGQG chain variableLEWIGQIYPGDGDTHYNGKFQGRVTITADKSTSTAYMELSSLRS region VH5EDTAVYYCSSSNWVGSYWGQGTLVTVSS (humanized) 13 5H10 LightDVVMTQTPLTLSVTIGQTASISCKSSQSLLESDGKTYLNWLSQR chain variablePGQSPKRLIYLVSRLDSGVPDRFTGSGSGTDFTLKISRVEAEDL region VK0GVYYCWQGTHLPQTFGGGTKLEIK (murine parent) 14 5H10 LightDVVMTQSPLTLSVTLGQPASISCKSSQSLLESDGKTYLNWLQQR chain variablePGQSPRRLIYLVSRLDSGVPDRFTGSGSGTDFTLKISRVEAEDV region VK1GVYYCWQGTHLPQTFGGGTKVEIK (humanized) 15 5H10 LightDVVMTQSPLSLPVTLGQPASISCKSSQSLLESDGKTYLNWLQQR chain variablePGQSPRRLIYLVSRLDSGVPDRFTGSGSGTDFTLKISRVEAEDV region VK2GVYYCWQGTHLPQTFGGGTKVEIK (humanized) 16 5H10 LightDVVMTQSPLSLPVTLGQPASISCKSSQSLLESDGKTYLNWLQQR chain variablePGQSPRRLIYLVSRLDSGVPDRFSGSGSGTDFTLKISRVEAEDV region VK3GVYYCWQGTHLPQTFGGGTKVEIK (humanized) 17 5H10 LightDVVMTQSPLSLPVTLGQPASISCKSSQSLLESDGKTYLNWFQQR chain variablePGQSPRRLIYLVSRLDSGVPDRFSGSGSGTDFTLKISRVEAEDV region VK4GVYYCWQGTHLPQTFGGGTKVEIK (humanized) 37 5H10 VH5 QIYPGDGDTHYNGKFQG CDR2

The skilled person will understand that the variable heavy and variablelight chains may be independently selected, or mixed and matched, fromthe antibodies provided herein. Thus, in some embodiments, theantibodies and fragments thereof provided herein comprise heavy andlight chain combinations selected from the group consisting of VH0/VK0,VH0/VK1, VH0/VK2, VH0/VK3, VH0/VK4, VH1/VK0, VH1/VK1, VH1/VK2, VH1/VK3,VH1/VK4, VH2/VK0, VH2/VK1, VH2/VK2, VH2/VK3, VH2/VK4, VH3/VK0, VH3/VK1,VH3/VK2, VH3/VK3, VH3/VK4, VH4/VK0, VH4/VK1, VH4/VK2, VH4/VK3, VH4/VK4,VH5/VK0, VH5/VK1, VH5/VK2, VH5/VK3, and VH5/VK4.

In some embodiments, the present disclosure provides antibodies orfragments comprising amino acid sequences having at least 80%, at least85%, at least 90%, at least 95%, or at least 99% homology to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 7-12. Insome embodiments, the present disclosure provides antibodies orfragments thereof comprising a heavy chain variable region according toa sequence selected from the group consisting of SEQ ID NOs: 7-12. Insome embodiments, the present disclosure provides antibodies orfragments comprising amino acid sequences having at least 80%, at least85%, at least 90%, at least 95%, or at least 99% homology to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 7-11,wherein the antibody or fragment comprises a heavy chain CDR1, CDR2, andCDR3 identical to SEQ ID NOs: 1, 2, and 3, respectively. In someembodiments, the present disclosure provides antibodies or fragmentscomprising amino acid sequences having at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% homology to an amino acidsequence of SEQ ID NO: 12, wherein the antibody or fragment comprises aheavy chain CDR1, CDR2, and CDR3 identical to SEQ ID NOs: 1, 37, and 3,respectively.

In some embodiments, the present disclosure provides antibodies orfragments comprising amino acid sequences having at least 80%, at least85%, at least 90%, at least 95%, or at least 99% homology to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 13-17.In some embodiments, the present disclosure provides antibodies orfragments thereof comprising a light chain variable region according toa sequence selected from the group consisting of SEQ ID NOs: 13-17. Insome embodiments, the present disclosure provides antibodies orfragments comprising amino acid sequences having at least 80%, at least85%, at least 90%, at least 95%, or at least 99% homology to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 13-17,wherein the antibody or fragment comprises a light chain CDR1, CDR2, andCDR3 identical to SEQ ID NOs: 4, 5, and 6, respectively.

In some embodiments, the present disclosure provides antibodies orfragments comprising amino acid sequences having at least 80%, at least85%, at least 90%, at least 95%, or at least 99% homology to: SEQ ID NO:7 and SEQ ID NO: 13; SEQ ID NO: 7 and SEQ ID NO: 14; SEQ ID NO: 7 andSEQ ID NO: 15; SEQ ID NO: 7 and SEQ ID NO: 16; SEQ ID NO: 7 and SEQ IDNO: 17; SEQ ID NO: 7 and SEQ ID NO: 13; SEQ ID NO: 7 and SEQ ID NO: 14;SEQ ID NO: 7 and SEQ ID NO: 15; SEQ ID NO: 7 and SEQ ID NO: 16; SEQ IDNO: 7 and SEQ ID NO: 17; SEQ ID NO: 8 and SEQ ID NO: 13; SEQ ID NO: 8and SEQ ID NO: 14; SEQ ID NO: 8 and SEQ ID NO: 15; SEQ ID NO: 8 and SEQID NO: 16; SEQ ID NO: 8 and SEQ ID NO: 17; SEQ ID NO: 9 and SEQ ID NO:13; SEQ ID NO: 9 and SEQ ID NO: 14; SEQ ID NO: 9 and SEQ ID NO: 15; SEQID NO: 9 and SEQ ID NO: 16; SEQ ID NO: 9 and SEQ ID NO: 17; SEQ ID NO:10 and SEQ ID NO: 13; SEQ ID NO: 10 and SEQ ID NO: 14; SEQ ID NO: 10 andSEQ ID NO: 15; SEQ ID NO: 10 and SEQ ID NO: 16; SEQ ID NO: 10 and SEQ IDNO: 17; SEQ ID NO: 11 and SEQ ID NO: 13; SEQ ID NO: 11 and SEQ ID NO:14; SEQ ID NO: 11 and SEQ ID NO: 15; SEQ ID NO: 11 and SEQ ID NO: 16;SEQ ID NO: 11 and SEQ ID NO: 17; SEQ ID NO: 12 and SEQ ID NO: 13; SEQ IDNO: 12 and SEQ ID NO: 14; SEQ ID NO: 12 and SEQ ID NO: 15; SEQ ID NO: 12and SEQ ID NO: 16; or SEQ ID NO: 12 and SEQ ID NO: 17.

In particular embodiments, the antibodies and fragments thereof compriseheavy and light chain combinations selected from the group consisting ofVH1/VK1, VH1/VK2, VH1/VK3, VH2/VK1, VH2/VK2, VH2/VK3, VH3/VK1, VH3/VK2,VH3/VK3, VH4/VK1, VH4/VK2, VH4/VK3, VH5/VK1, VH5/VK2, and VH5/VK3. Insome embodiments, the antibodies comprise an amino acid sequence havingat least 80%, at least 85%, at least 90%, at least 95%, or at least 99%sequence identity to SEQ ID NO: 8 or SEQ ID NO: 9; and an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or at least 99% sequence identity to SEQ ID NO: 16. In some embodiments,the antibody, or fragment thereof, comprises an amino acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 9; and anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, or at least 99% sequence identity to SEQ ID NO: 16; whereinthe antibody or fragment comprises a heavy chain CDR1, CDR2, and CDR3identical to SEQ ID NOs: 1, 2, and 3, respectively, and a light chainCDR1, CDR2, and CDR3 identical to SEQ ID NOs: 4, 5, and 6, respectively.In some embodiments, the antibody, or fragment thereof, comprises anamino acid sequence having at least 95% or at least 99% sequenceidentity to SEQ ID NO: 8 or SEQ ID NO: 9; and an amino acid sequencehaving at least 95% or at least 99% sequence identity to SEQ ID NO: 16;wherein the antibody or fragment comprises a heavy chain CDR1, CDR2, andCDR3 identical to SEQ ID NOs: 1, 2, and 3, respectively, and a lightchain CDR1, CDR2, and CDR3 identical to SEQ ID NOs: 4, 5, and 6,respectively. The antibody or fragment thereof may specifically bind toSCF248 but may not bind to SCF220. In some embodiments, the antibodiescomprise a heavy chain variable region according to SEQ ID NO: 8 and alight chain variable region according to SEQ ID NO: 16. In someembodiments, the antibodies comprise a heavy chain variable regionaccording to SEQ ID NO: 9 and a light chain variable region according toSEQ ID NO: 16.

In some embodiments, the antibodies and fragments provided hereincomprise a heavy chain variable region amino acid sequence according toSEQ ID NO: 7, 8, 9, 10, 11, or 12, or a variant thereof; and/or comprisea light chain variable region amino acid sequence according to SEQ IDNO: 13, 14, 15, 16, or 17, or a variant thereof. Variants may comprise1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions or deletions,or a combination thereof. In some embodiments, the amino acidsubstitutions are conservative substitutions. The anti-SCF antibodiesdisclosed herein having one or more amino acid substitution, insertion,deletion, or combination thereof in the CDR or variable light or heavychain region retain the biological activity of the correspondinganti-SCF antibody that does not have an amino acid substitution,insertion, or deletion relative to the sequences provided herein. Thus,the variant anti-SCF antibodies provided herein retain specific bindingto SCF248. The terms percent homology, sequence identity, sequencehomology, and the like are used interchangeably herein and refer to thenumber of identical amino acid sequences shared by two referencesequences, divided by the total number of amino acid positions,multiplied by 100.

In some embodiments, the present invention provides antibodies that bindto the same epitope as any one of the exemplary antibodies disclosedherein. Thus, in some embodiments, the present invention providesantibodies that compete for binding to SCF with the exemplary antibodiesprovided herein. For example, in some embodiments, the presentdisclosure provides antibodies that specifically bind to a region of theamino acid sequence provided herein as SEQ ID NO: 29. In someembodiments, antibodies provided herein specifically bind to an epitopecomprising the amino acid sequence of SEQ ID NO: 33 (ASSLRNDSSSSNRK) orSEQ ID NO: 36 ASSLRNDSSSSNR). In some embodiments, the presentdisclosure provides antibodies that specifically bind to an epitopeconsisting of an amino acid sequence according to SEQ ID NO: 33 or SEQID NO: 36. In some embodiments, the present disclosure providesantibodies that specifically bind to an epitope comprising at least 5,at least 6, at least 7, at least 8, at least 9, or at least 10contiguous amino acids of SEQ ID NO: 33.

In some embodiments, the antibodies and fragments thereof providedherein comprise the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3 ofthe heavy and light chain variable regions provided herein, or variantsthereof. Thus, in some embodiments, the antibodies and fragments thereofprovided herein include antibodies wherein the HCDRs are the HCDRs ofSEQ ID NO: 7, 8, 9, 10, 11, or 12; and/or wherein the LCDRs are theLCDRs of SEQ ID NOs: 13, 14, 15, 16, or 17. For example, in someembodiments, the antibodies and fragments thereof comprise amino acids31-35, 50-65, and 95-102 of any one of the heavy chain variable regionsprovided herein, as defined by the Kabat numbering scheme. In someembodiments, the antibodies and fragments thereof comprise amino acids24-34, 50-56, and 89-97 of any one of the light chain variable regionsprovided herein, as defined by the Kabat numbering scheme.

Exemplary humanized antibodies are provided herein. Additional anti-SCFantibodies comprising the heavy and light chain CDRs provided herein, orvariants thereof, may be generated using any human framework sequence,and are also encompassed in the present invention. In one embodiment,framework sequences suitable for use in the present invention includethose framework sequences that are structurally similar to the frameworksequences provided herein. Further modifications in the frameworkregions may be made to improve the properties of the antibodies providedherein. Such further framework modifications may include chemicalmodifications; point mutations to reduce immunogenicity or remove T cellepitopes; or back mutation to the residue in the original germlinesequence.

In some embodiments, such framework modifications include thosecorresponding to the mutations exemplified herein, includingbackmutations to the germline sequence. For example, in one embodiment,one or more amino acids in the human framework regions of the VH and/orVL of the humanized antibodies provided herein are back mutated to thecorresponding amino acid in the parent murine antibody. The presentinvention also encompasses humanized antibodies that bind to SCF (e.g.,SCF248) and comprise framework modifications corresponding to theexemplary modifications described herein with respect to any suitableframework sequence, as well as other framework modifications thatotherwise improve the properties of the antibodies. In otherembodiments, the antibodies provided herein comprise one or moremutations to improve stability, improve solubility, alter glycosylation,and/or reduce immunogenicity, such as, for example, by targeted aminoacid changes that reduce deamidation or oxidation, reduce isomerization,optimize the hydrophobic core and/or charge cluster residues, removehydrophobic surface residues, optimize residues involved in theinterface between the variable heavy and variable light chains, and/ormodify the isoelectric point.

The anti-SCF antibodies and fragments thereof provided herein mayfurther comprise Fc region modifications to alter effector functions. Fcmodifications may be amino acid insertions, deletions, or substitutions,or may be chemical modifications. For example, Fc region modificationsmay be made to increase or decrease complement binding, to increase ordecrease antibody-dependent cellular cytoxicity, or to increase ordecrease the half-life of the antibody. Some Fc modifications increaseor decrease the affinity of the antibody for an Fcγ receptor such asFcγRI, FcγRII, FcγRIII, or FcRn. Various Fc modifications have beendescribed in the art, for example, in Shields et al., J Biol. Chem 276;6591 (2001); Tai et al. Blood 119; 2074 (2012); Spiekermann et al. JExp. Med 196; 303 (2002); Moore et al. mAbs 2:2; 181 (2010);Medzihradsky Methods in Molecular Biology 446; 293 (2008); Mannan et al.Drug Metabolism and Disposition 35; 86 (2007); and Idusogie et al. JImmunol 164; 4178 (2000). In some embodiments, Fc region glycosylationpatters are altered. In other embodiments, the Fc region is modified bypegylation (e.g., by reacting the antibody or fragment thereof withpolyethylene glycol (PEG). Exemplary Fc modifications includemodifications at one or more amino acid position selected from the groupconsisting of 228, 233, 234, 235, 236, 241, 248, 265, 297, 309, 331, and409 (Kabat numbering; Kabat et al., Sequences of Immunological Interest,Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). Inembodiments, the antibody has modifications to reduce or abolisheffector function. In embodiments, the antibody is an IgG1 antibodyhaving one or more Fc modification selected from the group consisting ofE233P, L234V, L234A, L235V, L235A, G236 (deleted), D265A, D270A, N297Aand N297Q. In embodiments, the antibody is an IgG4 antibody having oneor more Fc modification selected from the group consisting of S228P,E233P, F234A, F234V, L235A, L235V, S241P, L248E, D265A, D265T, L309L,and R409K. In embodiments, the anti-SCF antibodies provided hereincomprise a S241P mutation and an L248E mutation.

In embodiments, the present disclosure provides antibodies providedherein that comprise a human IgG4 constant region according to SEQ IDNOs: 40 and 41. In embodiments, the present disclosure providesantibodies comprising at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, or about 99% sequence identity to SEQ IDNO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In embodiments, thepresent disclosure provides antibodies comprising a heavy chainaccording to SEQ ID NO: 40 and a light chain according to SEQ ID NO: 41.In embodiments, the present disclosure provides antibodies comprising aheavy chain according to SEQ ID NO: 42, 43, 44, 45, or 46 and a lightchain according to SEQ ID NO: 47, 48, 49, or 50. In embodiments, thepresent disclosure provides an antibody comprising a heavy chainaccording to SEQ ID NO: 42 and a light chain according to SEQ ID NO: 49.In embodiments, the present disclosure provides an antibody comprising aheavy chain according to SEQ ID NO: 43 and a light chain according toSEQ ID NO: 49. In embodiments, the present disclosure provides anantibody comprising a heavy chain according to SEQ ID NO: 44 and a lightchain according to SEQ ID NO: 49. In embodiments, the present disclosureprovides an antibody comprising a heavy chain according to SEQ ID NO: 45and a light chain according to SEQ ID NO: 49. In embodiments, thepresent disclosure provides an antibody comprising a heavy chainaccording to SEQ ID NO: 46 and a light chain according to SEQ ID NO: 49.

In some embodiments, the antibodies provided herein are specific forSCF248 and do not bind to SCF220. Thus, the antibodies provided hereinare capable of specifically inhibiting the interaction between SCF248and c-Kit that induces and perpetuates chronic inflammatory responsesand fibrosis in inflammatory and fibrotic renal diseases. Moreover, theantibodies provided herein are capable of specifically inducing theinternalization of SCF and thereby reducing the interaction betweenSCF248 and c-Kit. Accordingly, in some embodiments the presentdisclosure provides methods for treating inflammatory and fibrotic renaldiseases comprising administering to patients in need thereof antibodiesthat are specific for SCF248, and are safe and effective in variousinflammatory and fibrotic renal diseases discussed herein and known inthe art.

For preparation of monoclonal antibodies, any technique that providesfor the production of antibody molecules by continuous cell lines inculture may be used (see e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).These include, but are not limited to, the hybridoma techniqueoriginally developed by Kohler and Milstein and the trioma technique,the human B-cell hybridoma technique (See, e.g., Kozbor et al., Immunol.Today, 4:72 (1983)), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., in Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96 (1985)). Alternatively, theantibodies may be made by recombinant DNA methods. In some embodiments,antibodies in accordance with the present disclosure may be made byisolating monoclonal antibodies from phage display libraries using thetechniques described, for example, in Clackson et al., Nature 352:624-28(1991) and Marks et al., J. Mol. Biol. 222(3):581-97 (1991). In someembodiments, the antibodies are fully human antibodies constructed bycombining Fv clone variable domain sequence(s) selected fromhuman-derived phage display or yeast display libraries with known humanconstant domain sequence(s).

In some embodiments provided herein, the antibodies are prepared from ahybridoma. Using the hybridoma method, a mouse, hamster, or otherappropriate host animal, is immunized by injecting an immunizing peptideto elicit the production by lymphocytes of antibodies that willspecifically bind to an immunizing antigen. Alternatively, lymphocytescan be immunized in vitro. Following immunization, the lymphocytes areisolated and fused with a suitable myeloma cell line using, for example,polyethylene glycol, to form hybridoma cells that can then be selectedaway from unfused lymphocytes and myeloma cells. Hybridomas that producemonoclonal antibodies directed specifically against a chosen antigen asdetermined by immunoprecipitation, immunoblotting, or by an in vitrobinding assay such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA) can then be propagated in vitro (e.g., inculture) using standard methods (Goding, Monoclonal Antibodies:Principles and Practice, Academic Press, 1986) or in vivo as ascitestumors in an animal. The monoclonal antibodies can then be purified fromthe culture medium or ascites fluid as described for polyclonalantibodies above.

In some embodiments, the antibodies provided herein are generated usingthe murine hybridoma system. Hybridoma production in the mouse is awell-established procedure. Immunization protocols and techniques forisolation of immunized splenocytes for fusion are known in the art.Fusion partners (e.g., murine myeloma cells) and fusion procedures arealso known. Embodiments of the technology herein provide antibodies(e.g., monoclonal antibodies) produced from a hybridoma prepared byimmunizing mice with a peptide that is a portion or fragment of the SCFprotein.

In some embodiments, the antibodies specific for SCF248 provided hereinare generated by immunizing mice with a peptide having an amino acidsequence that is largely or exclusively within exon 6. For example, theimmunizing peptide comprises any stretch of 5 or more amino acids withinSEQ ID NO: 34. As another example, the immunizing peptide comprises anystretch of 5 or more amino acids beginning at amino acid position 20 ofSEQ ID NO: 29. As another example, the immunizing peptide comprises astretch of 5 or more amino acids beginning at amino acid position 20 ofSEQ ID NO: 29 and ending at any one of positions 25 to 38 of SEQ ID NO:29. Thus, in some embodiments, the immunizing peptide comprises theamino acid sequence of exon 6 after the cleavage site, and is eitherfully contained within exon 6 or comprises only 1, 2, 3, 4, or 5 aminoacids of exon 7. In some embodiments, the immunizing peptide comprisesor consists of SEQ ID NO: 30. In some embodiments, the immunizingpeptide comprises any of the peptides provided herein or conservativevariants thereof. Conservative variants may comprise 1, 2, 3, 4, or 5amino acid substitutions or deletions, or a combination thereof. Asprovided above, in some embodiments, the antibodies generated using theimmunizing peptides provided herein have an epitope that falls entirelyor largely within exon 6. By “largely within” it is meant that at least75%, at least 80%, at least 85%, at least 90%, or at least 95% of thepeptide falls within exon 6. In some embodiments, the epitope begins atthe cleavage site of exon 6 (i.e., between the alanines at amino acidpositions 19 and 20 of SEQ ID NO: 29 and extends to the end of exon 6.In some embodiments, the epitope begins at the cleavage site of exon 6and extends to the 1^(st), 2^(nd), 3^(rd), 4^(th) or 5^(th) n-terminalamino acid of the transmembrane domain. In some embodiments, the epitopecomprises or consists of SEQ ID NO: 33. In some embodiments, theantibody referred to herein as 5H10 (including the murine, chimeric, andhumanized 5H10 antibodies) binds to an epitope of SCF comprising orconsisting of SEQ ID NO: 33.

In some embodiments, methods provided herein were used to generateantibodies referred to herein as 5H10. In some embodiments, the antibody“5H10” is also referred to herein as “OpSCF.” Antibody 5H10advantageously binds SCF248 with high specificity and does not bindSCF220. The amino acid sequences of the murine parent antibody 5H10, aswell as humanized variants thereof, are provided herein (see, Table 1).

In one embodiment, the present invention provides methods of use ofbispecific or multispecific antibodies specific for SCF and at least oneother antigen or epitope. The anti-SCF antibodies and fragments thereofprovided herein may be tested for binding to SCF using the bindingassays provided herein, or any other binding assay known in the art.

Unless otherwise stated, the practice of the present invention employsconventional molecular biology, cell biology, biochemistry, andimmunology techniques that are well known in the art and described, forexample, in Methods in Molecular Biology, Humana Press; MolecularCloning: A Laboratory Manual, second edition (Sambrook et al., 1989),Current Protocols in Immunology (J. E. Coligan et al., eds., 1991);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal antibodies: a practical approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); Phagedisplay: a laboratory manual (C. Barbas III et al, Cold Spring HarborLaboratory Press, 2001); and Using antibodies: a laboratory manual (E.Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999).

Methods of Treatment

As used herein, the terms “treatment” or “treating” refers to boththerapeutic treatment and prophylactic or preventive measures. Subjectsin need of treatment include those subjects that already have thedisease or condition, as well as those that may develop the disease orcondition and in whom the object is to prevent, delay, or diminish thedisease or condition. As used herein, the term “subject” denotes amammal, such as a rodent, a feline, a canine, and a primate. Preferably,a subject according to the invention is a human. The term“therapeutically effective amount,” as used herein, refers to the amountof a compound or composition that is necessary to provide a therapeuticand/or preventative benefit to the subject.

In one aspect the present invention provides methods for treating asubject for an inflammatory and/or fibrotic renal disease. In oneaspect, the present disclosure provides antibodies that are for use as amedicament useful for treating renal diseases, such as inflammatoryand/or fibrotic renal diseases. In one aspect, the present disclosureprovides antibodies for use in a method of treatment of a renal diseasesuch as an inflammatory and/or fibrotic renal disease. In someembodiments, the inflammatory renal disease is a chronic inflammatoryrenal disease. Exemplary inflammatory and/or fibrotic renal diseasesinclude, for example, renal fibrosis, renal cirrhosis, Interstitialfibrosis and tubular atrophy (IFTA) of the kidney, chronic kidneydisease, end stage renal disease (ESRD), Goodpasture's syndrome,glomerulonephritis, membranoproliferative glomerulonephritis (MPGN),chronic renal allograft rejection, nephrogenic systemic fibrosis, andnephropathy (e.g., IgA nephropathy, focal segmental glomerulosclerosis,rapidly progressive glomerulonephritis, crescentic glomerulonephritis,lupus nephritis, hypertensive nephropathy, or diabetic nephropathy).

In some embodiments, the antibodies and fragments thereof disclosedherein may be administered to the subject by at least one route selectedfrom parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracerebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intratympanic, intrauterine, intravesical,intravitreal, bolus, subconjunctival, oral, vaginal, rectal, buccal,sublingual, intranasal, intratumoral, and transdermal.

In embodiments, the antibodies and fragments thereof disclosed hereinmay be administered to a subject in need thereof in combination with oneor more additional therapy. The one or more additional therapy may be aprocedure such as a surgical procedure or dialysis, or may be atherapeutic agent, such as an agent designed to mitigate or reducesymptoms of a disease or disorder associated with renal fibrosis and/orinflammation.

The present invention is further illustrated by reference to thefollowing Examples. However, it should be noted that these Examples,like the embodiments described above, are illustrative and are not to beconstrued as restricting the scope of the invention in any way.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the disclosure and are not meant to limit the presentdisclosure in any fashion. Changes therein and other uses which areencompassed within the spirit of the disclosure, as defined by the scopeof the claims, will be recognized by those skilled in the art.

An overview of the tissue injury/disease process is summarized inFIG. 1. A disease process initiates inflammation. c-Kit+ immune cellsproduce cytokines that cause fibroblasts to change into activatedmyofibroblasts which express SCF248 on their surface. The expression ofSCF248 on the surface of myofibroblasts and other cells activates moreimmune cells, resulting in cytokine release of IL-4, IL-9, IL-13, IL-25,TGFβ, and other cytokines, perpetuating inflammation. Myofibroblastssecrete extracellular matrix proteins, collagen, and fibronectin,leading to fibrosis diseases such as chronic kidney disease and others.An exemplary mechanism of an antibody of the instant disclosure whichtargets SCF248 in glomerulonephritis (GN) as an exemplary renal disease(said antibody referred to herein as OpSCF and/or as 5H10) is summarizedin FIG. 2.

As provided above, SCF has two isoforms which result from alternativesplicing: SCF248 and SCF220. SCF248 and SCF220 differ by exon 6. SCF220is associated with homeostatic functions, and SCF248 is associated withinflammation and fibrosis. SCF248 activates immune cells duringinflammation and is sometimes called “soluble SCF.” SCF248 is expressedon various cell types including myofibroblasts, activated epithelia,endothelia, macrophages, eosinophils, mast cells, and monocytes (FIG.3). The SCF248 isoform results in cleavage of monomeric cleavedextracellular domain, called SCF165. The amino acid sequence of exon 6is provided herein as SEQ ID NO: 34.

Example 1: Production of Anti-SCF mAbs Utilizing Hybridoma Technology

A peptide comprising ASSLRNDSSSSNRKAKNPPGD (SEQ ID NO: 30) was used togenerate antibodies that bind to SCF248. The immunization peptidecomprised a portion of exon 6, i.e. the SCF248 isoform of stem cellfactor. In particular, the immunization peptide comprised a portion ofexon 6 that begins after a cleavage site as defined herein. Mice wereimmunized with a peptide according to SEQ ID NO: 30 with a standardprotocol. The determination of high titer serum antibodies indicated theappropriate immunization and fusion hybridomas were made. Culturesupernatants were analyzed from individual clones for SCF-specificantibodies and chosen based upon specificity. Hybridomas producingspecific monoclonal antibodies against the peptide were propagated andthe monoclonal with the highest titer was subsequently tested inbiologically relevant cultures. Antibody 5H10 had high specificity forSCF248 and no cross-reactivity with SCF220. No other monoclonalantibodies produced by the hybridomas had high specificity for SCF248without cross-reactivity with SCF220. Thus, 5H10 was selected forfurther characterization, development, and chimerization and subsequenthumanization.

Example 2. 5H10 Binding to SCF248 Complete Extracellular Domain

The murine 5H10 antibody obtained as described in Example 1 was directlyconjugated with a fluorescent marker and the labeled antibody wasincubated with Sl/S14 hSCF248 cells, which express SCF248; Sl/S14hSCF220 cells, which express SCF220; or control cells that do notexpress SCF. Binding of the labeled antibody to the cells was assessedby flow cytometry. The specificity of 5H10 for SCF248 and lack ofcrossreactivity with SCF220 is shown in FIG. 4A.

Binding of the murine 5H10 antibody to the cleaved extracellular domain(ECD) containing only amino acids 1-165 of SCF, vs the complete ECDcontaining amino acids 1-194 of SCF, was assessed by an ELISA method.The antibody bound to the complete SCF ECD but not to the cleaved SCFECD (FIG. 4B), demonstrating that the antibody is specific for thecomplete extracellular domain and does not bind to the monomeric cleavedECD that circulates in blood.

To assess the ability of 2G8 and 5H10 antibodies to internalize SCF248on myofibroblasts, antibodies were labeled with pHrodo red, which iscolorless at neutral pH and fluoresces red at the low pH within anendosome. Labeled antibodies were incubated with cultured human IPFmyofibroblasts for 45 minutes and red fluorescence was visualized bymicroscopy. As shown in FIG. 5, the dye-labeled antibodies, but notcontrol IgG, were rapidly internalized. 5H10 was internalized morerapidly and resulted in higher fluorescence compared to 2G8.

SCF triggers c-kit to signal by two distinct pathways: the MEK/ERKpathway and the PI3K/AKT pathway. A study was conducted to determinewhether the murine 5H10 antibody inhibits intracellular signaling inc-kit positive cells in either or both of these pathways. Eosinophilswere incubated with SCF248-expressing cell lines, in the presence ofeither 5H10 or IgG control, and phospho-protein expression was measuredwith a BioRad Bio-Plex assay system. 5H10 significantly decreased thephospho-MEK and phosphor-AKT levels, indicating that the antibodysignificantly reduced c-kit mediated intracellular signaling (FIG. 6).

Taken together, the results of these studies indicated that antibody5H10 binds specifically to and internalizes SCF248, and does notcross-react with the SCF220 isoform or the cleaved ECD. Moreover, 5H10significantly inhibits the intracellular signaling pathways in c-kitpositive cells that perpetuate inflammation.

Example 3. Humanized 5H10

Chimeric antibodies derived from 5H10 were produced by subcloning thevariable domains of the heavy and light chains into a vector with ahuman IgG4 backbone. Chimeric antibodies were expressed and purifiedusing standard protocols. 2G8 is a previously developed antibody thatbinds to SCF248 and SCF220, and contains a lambda light chain. Thechimeric heavy and light chains of 2G8 were named VH0 and VL0,respectively. 5H10, the SCF248-specific antibody provided herein,contains a kappa light chain. The chimeric heavy and light chains of5H10 were named VH0 and VK0, respectively.

The chimeric antibodies were humanized. Humanized heavy chains retainedthe same complementarity-determining regions (CDRs) but more“human-like” framework regions, and several humanized variants of eachof 2G8 and 5H10 variable heavy chains, referred to herein as VH1, VH2,VH3, VH4, and VH5, were generated. Humanized kappa light chain variantsof 5H10, referred to herein as VK1, VK2, VK3, and VK4, were alsogenerated. Humanized lambda light chains of 2G8 were named VL1, VL2,VL3, and VL4. The 2G8 and 5H10 combinations of chimeric and humanizedlight chains and heavy chains tested are shown in Table 2 and Table 3,respectively. As shown in Table 2, certain heavy and light chaincombinations of the 5H10 antibody variants resulted in high binding tohSCF248.

TABLE 2 Binding score for 2G8 chimeric and humanized clones to S1/S14hSCF248 cells 2G8 mAb Binding Score VH0/VL0 high VH1/VL1 Moderate highVH1/VL3 moderate VH1/VL4 Moderate VH2/VL1 Moderate VH2/VL4 ModerateVH3/VL1 Moderate VH3/VL4 Moderate VH4/VL1 Moderate VH4/VL2 ModerateVH5/VL1 Moderate high VH5/VL4 Moderate

TABLE 3 Binding score for 5H10 chimeric and humanized clones to S1/S14hSCF248 cells 5H10 mAb Binding Score VH0/VK0 high VH1/VK1 High VH1/VK2High VH1/VK3 High VH1/VK4 No binding VH2/VK2 Moderate high VH2/VK3 highVH3/VK2 Moderate VH3/VK3 Moderate VH4/VK2 Moderate low VH4/VK3 Moderatelow VH5/VK2 Low VH5/VK3 low

Binding affinity was also assessed using a BiaCore analysis. BiaCoredata showed that the affinity for immobilized SCF248 peptide antigen ofall humanized 5H10 antibodies having the VK1, VK2, or VK3 light chainwas very similar to the binding affinity of the parental murine 5H10using this assay. Humanized 5H10 antibodies having a VK4 light chain didnot bind to the peptide.

TABLE 4 Biacore data VK0 VK1 VK2 VK3 VK4 VH0 1.00 0.91 VH1 0.92 0.990.97 0.94 — VH2 0.98 0.99 0.94 — VH3 1.15 1.16 1.10 — VH3 1.27 1.32 0.85— VH5 1.07 0.93 1.09 —

5H10 clones VH1/VK3, VH12/VK3, VH13/VK3, VH14/VK3, and VH15/VK3 wereassessed by flow cytometry for binding to the SCF248-expressing cellline. As shown in FIGS. 7A and 71B, VH1/VK3 and VH12/VK3 exhibited highbinding, maximized at 1 μg/mL. The negative control was secondaryantibody only. No binding was observed with the controlSCF220-expressing cell line (not shown).

Example 4. In Vitro Blockade of the Interaction of SCF and c-Kit

The humanized 5H10 antibodies were tested for their capacity to inhibitthe SCF-c-kit interaction and the inflammation feed-forward loop invitro. Cultured human IPF myofibroblasts (Mfb), which express surfaceSCF248, were overlaid with LAD2 mast cells, an SCF-responsive cell line.Absent any other intervention, the Mfb stimulate the LAD2 cells, whichproduce cytokines to stimulate Mfb to produce additional cytokines andextracellular matrix proteins. In this assay, the readout forinflammation and the feed-forward loop is mRNA for CCL11, collagens 1and 3, and fibronectin.

Murine 5H10 and humanized (VH1/VK3, VH2/VK3, VH3/VK3, VH4/VK3, andVH5/VK3) 5H10 antibodies were pre-incubated with Mfb at concentrationsof 1 μg/mL and 10 μg/mL to assess their capacity to inhibit thefeed-forward loop. Results are shown in FIGS. 8A-8D. The humanizedVH1/VK3 antibody consistently demonstrated inhibition of the SCF—c-kitinteraction, even at the lower concentration.

Example 5. Correlation Between SCF248 in Kidney Biopsies and DiseaseProgression

RNAseq was performed to quantitate SCF248 mRNA from kidney biopsies frompatients with focal segmental glomerulosclerosis (FSGS). A significantinverse correlation of SCF248 mRNA with glomerular filtration rate wasobserved as shown in FIG. 9A. A positive correlation of SCF248 mRNA withthe % interstitial fibrosis was also observed, as shown in FIG. 9B.Further, a positive correlation of SCF248 mRNA with the percentage ofmononuclear white blood cells in the kidney biopsy was identified, asshown in FIG. 9C. Together, these data suggest an association of SCF248with markers of inflammation and progressive renal insufficiency.

Moreover, a chronic kidney disease subject's plasma level of cleavedstem cell factor extracellular domain, SCF165, was significantlyinversely correlated with estimated glomerular filtration rate (eGFR)(FIG. 10); and correlated with the urinary albumin/creatinine ratio(UACR) (FIG. 11), in chronic kidney disease.

Example 6. 5H10 Effectively Treats Disease in a Model of Chronic KidneyDisease

In areas of human renal fibrosis, SCF248 is expressed. In comparison tosamples stained by immunohistochemistry with control IgG (FIG. 12A),samples stained with murine 5H10 antibody were strongly positive forSCF248 in the tubulo-interstitium (FIG. 12B and FIG. 12C). Additionally,staining for mast cell tryptase revealed the presence of mast cells inthe kidneys of patients with diabetic nephropathy and IgA nephropathy(FIG. 13B and FIG. 13C), but not in a healthy patient's kidney. (FIG.13A).

A mouse model of chronic kidney disease (CKD) was used to investigatethe effect of 5H10 on disease progression and survival. C57/Black 6TGFβ1 transgenic mice (TGFβ mice) overexpress TGFβ1 in the liver underthe control of the albumin promoter, which leads to an increase incirculating TGFβ that promotes tissue fibrosis. TGFβ mice experienceprogressive glomerular and mesangial expansion and decreased podocytedensity. Progressive interstitial fibrosis results in loss of kidneyweight and death.

TGFβ mice were given either 5H10 or control antibody for four weeksstarting at two weeks of age at a dose of 20 mg/kg, twice a week. Theexperiment was terminated at week six due to mortality. In comparison tomice treated with control IgG antibody, mice treated with 5H10 had asignificantly improved survival (p=0.03) and a reduced loss in kidneyweight (p=0.03) (FIG. 14A and FIG. 14B). The kidneys were scored in amasked fashion by an experienced nephropathologist, and the kidneys inthe 5H10-treated group had significantly less fibrosis (FIG. 14C).

In order to evaluate kidney histology during disease progression, TGFβmice were dosed at 5 mg/kg, twice a week with 5H10 or control antibodystarting at two weeks of age and autopsied after two weeks during CKDdisease progression. In comparison to control IgG, glomerular volume andmesangial volume increased less with 5H10 treatment, suggesting lesstissue damage. Early in the course of CKD, glomerular volume andmesangial volume increase with the influx of inflammatory cells and theextracellular matrix. The podocyte density was maintained with 5H10therapy, indicating less podocyte dropout and less glomerular swelling(FIG. 15A, FIG. 15B, FIG. 15C). RNA sequencing demonstratedstatistically significant decreases in matrix proteins Collagen Type 3Alpha 1 chain (FIG. 16A), Collagen Type 6 Alpha 3 chain (FIG. 16B),Collagen Type XV Alpha 1 chain (FIG. 16C), Fibronectin Type III Domaincontaining 1 (FIG. 16D), Fibulin 1 (FIG. 16E), andMicrofibril-associated protein 4 (FIG. 16F), in animals treated withm5H10. Thus, the administration of 5H10 antibody in a model of chronickidney disease significantly reduced kidney fibrosis and improvedsurvival, indicating that the antibody is useful as a treatment forkidney disease.

Publications, patents and patent applications cited herein arespecifically incorporated by reference in their entireties. While thedescribed invention has been described with reference to the specificembodiments thereof it should be understood by those skilled in the artthat various changes may be made and equivalents may be substitutedwithout departing from the true spirit and scope of the invention. Inaddition, many modifications may be made to adopt a particularsituation, material, composition of matter, process, process step orsteps, to the objective spirit and scope of the described invention. Allsuch modifications are intended to be within the scope of the claimsappended hereto.

1. A method for treating an inflammatory or fibrotic renal disease in asubject in need thereof, the method comprising administering to thesubject an antibody or antigen binding fragment thereof thatspecifically binds to stem cell factor (SCF), wherein the antibody orantigen binding fragment thereof comprises heavy chain CDR1, CDR2, andCDR3 comprising SEQ ID NOs: 1, 2, and 3, respectively; and light chainCDR1, CDR2, and CDR3 according to SEQ ID NOs: 4, 5, and 6, respectively.2. The method of claim 1, wherein the antibody or fragment thereofcomprises a heavy chain variable region having at least 80% identity toa sequence selected from SEQ ID NOs: 7, 8, 9, 10, and
 11. 3. The methodof claim 1, wherein the antibody or fragment thereof comprises a heavychain variable region having at least 90% identity to a sequenceselected from SEQ ID NOs: 7, 8, 9, 10, and
 11. 4. The method of claim 1,wherein the antibody or fragment thereof comprises a light chainvariable region having at least 80% identity to a sequence selected fromSEQ ID NOs: 13, 14, 15, and
 16. 5. The method of claim 1, wherein theantibody or fragment thereof comprises a light chain variable regionhaving at least 90% identity to a sequence selected from SEQ ID NOs: 13,14, 15, and
 16. 6. The method of claim 1, wherein the antibody orfragment thereof comprises a heavy chain variable region amino acidsequence selected from SEQ ID NOs: 7, 8, 9, 10, and 11; and a lightchain variable region amino acid sequence selected from SEQ ID NOs: 13,14, 15, and
 16. 7. The method of claim 1, wherein the antibody orfragment thereof comprises a heavy chain variable region amino acidsequence according to SEQ ID NO: 7 and a light chain variable regionamino acid sequence according to SEQ ID NO:
 16. 8. The method of claim1, wherein the antibody or fragment thereof comprises a heavy chainvariable region amino acid sequence according to SEQ ID NO: 8 and alight chain variable region amino acid sequence according to SEQ ID NO:16.
 9. The method of claim 1, wherein the antibody or fragment thereofcomprises a heavy chain variable region amino acid sequence according toSEQ ID NO: 9 and a light chain variable region amino acid sequenceaccording to SEQ ID NO:
 16. 10. The method of claim 1, wherein theantibody or fragment thereof comprises a heavy chain variable regionamino acid sequence according to SEQ ID NO: 10 and a light chainvariable region amino acid sequence according to SEQ ID NO:
 16. 11. Themethod of claim 1, wherein the antibody or fragment thereof comprises aheavy chain variable region amino acid sequence according to SEQ ID NO:11 and a light chain variable region amino acid sequence according toSEQ ID NO:
 16. 12. The method of claim 1, wherein the antibody orfragment thereof is humanized.
 13. The method of claim 1, wherein theantibody is a monoclonal antibody.
 14. The method of claim 13, whereinthe antibody comprises a human IgG4 domain.
 15. The method of claim 14,wherein the IgG4 domain comprises a S241P mutation at amino acid residue241 and an L248E mutation at amino acid residue 248, wherein thenumbering of the residues is that of the Kabat numbering system.
 16. Themethod of claim 13, wherein the antibody comprises a heavy chainaccording to SEQ ID NO: 42 and a light chain according to SEQ ID NO: 49.17. The method of claim 13, wherein the antibody comprises a heavy chainaccording to SEQ ID NO: 43 and a light chain according to SEQ ID NO: 49.18. The method of claim 1, wherein the antibody or fragment thereofspecifically binds to SCF248.
 19. The method of claim 1, wherein theantibody does not bind to SCF220.
 20. The method of claim 1, wherein theinflammatory or fibrotic renal disease is selected from the groupconsisting of chronic kidney disease (CKD), end stage renal disease(ERSD), renal fibrosis, glomerulonephritis, and nephropathy.
 21. Themethod of claim 20, wherein the nephropathy or glomerulonephritis is IgAnephropathy, diabetic nephropathy, focal segmental glomerulosclerosis,rapidly progressive glomerulonephritis, crescentic glomerulonephritis,lupus nephritis, hypertensive nephropathy, or diabetic nephropathy.